Stem cells have the capacity to self-renew and to generate differentiated cells. Both adult and embryonic stem cells hold great potential for regenerative medicine, and gene therapy. Hematopoietic stem cells (HSCs) have been the most extensively studied and serve as a prototype model to define the general biological properties of stem cells. During embryogenesis, hematopoiesis is highly coordinated with angiogenesis. Recent studies have suggested that hematopoietic and endothelial precursors originate from a common ancestor, the hemangioblast. The key molecular players determining the fate of the hemangioblast are not fully elucidated. Receptor tyrosine kinases (RTKs), receiving and interpreting extracellular signals, play an important role in cell proliferation and differentiation. Endothelial cell receptor tyrosine kinase EphB4 (HTK) and its ligand, ephrinB2, are critical for angiogenesis. Loss of either EphB4 or ephrinB2 cause fatal abnormalities of capillary formation in null mice. We originally identified EphB4 from human bone marrow CD34+ cells. Our recent studies indicated that forced expression of EphB4 influence the function and phenotype of primitive human hematopoietic cells, enforcing preferential megakaryocytic and erythroid differentiation. This proposal seeks to further assess the role of EphB4 signaling in hematopoietic stem cells and the possibility that the EphB4 may be a common molecule bridging angiogenesis and hematopoiesis. In vitro assays of embryonic stem (ES) cells and in vivo experiments, including knockout mice and bone marrow transplantation, will be conducted to assess the differentiation potential of either genetically deficient or activated EphB4. Our preliminary data from in vitro differentiation of genetically engineered ES cells show that EphB4 signaling plays an important role in hemangioblasts and the early development of hematopoietic and endothelial cells. These studies identify EphB4 as one of the molecular modulators of hemangioblast formation. If successful, the results of this proposal will provide insight into the molecular mechanisms governing hemangioblasts and their differentiation along vascular and hematopoietic lineages. Manipulation of the signals that control hematopoiesis and vasculogenesis offer a broad spectrum of therapeutic potential for hematologic, oncologic and cardiovascular diseases. This Mentored Research Scientist Development Award will substantially advance the candidate's goal of developing an independent basic research program in an academic institution while contributing new training in stem cell biology and angiogenesis.